Mucus secretion is an important mechanism defending from irritants inhaled into the lungs during breathing. Secreted mucus forms a thin film of viscoelastic gel on the surface of the airways that protects the epithelial cells from irriants inhaled into the lungs by entrapping foreign debris, bacteria, and viruses and clearing them from the airway by ciliary movement; the whole process is termed mucociliary clearance. Besides its protective role, mucus could also have a pathologic roles in disease conditions, such as cystic fibrosis (CF), asthma, and chronic obstructive pulmonary disease (COPD), where excessive production of mucus (hypersecretion) and/or changes in its biophysical properties (viscoelasticity) result in the accumulation of thick, sticky mucus in the lungs, effectively impairing mucociliary clearance process. Since mucus is practically colorless viscous substance current video and light microscopy techniques used for studying mucus secretion suffer from poor resolution, sensitivity and limited temporal resolution. Fluorescence studies of mucin secretion are hampered by lack of well characterized fluorescent labels of mucin molecules and by difficulty to image in real-time a very rapid (~100 ms) secretion and swelling process. We recently tried a fluorescence approach with various fluorescence dyes to enable monitoring of mucin release on the cellular level and realized that the most interesting (important) processes occur in the initial steps when granules and generated mucus patches are small, frequently bellow optical resolution limits (<0.5 ?m). In this situation it is impossible to quantitatively reate fluorescence intensity changes (decreases) to mucus swelling. But one probe we tested (Acridine Orange - AO) exhibited very promising properties. The probe very effectively accumulates in low pH mucus granules inside the cell forming dimers/aggregates. The emission of the dimer/aggregate form is shifted 100 nm toward the red and its fluorescence lifetime is very long (over 10 times longer than fluorescence lifetime of the monomer). Also the brightness of aggregates is relatively high, making their detection easier. When mucus is released and swells, the equilibrium between monomers and aggregates quickly shifts toward monomers, producing a distinct change in color (from red to green). We expect this could be a great opportunity to develop/utilize the first fluorescence probe for studying the kinetics of mucus expansion. AO is one of the longest known fluorescent markers, and it was a surprise for us to realize that the amount of the information regarding the monomer-dimer/aggregates equilibrium is very limited. Multiple studies done over the past 50 years only give limited and partial information because of technical difficulties that were impossible to overcome with the available technologies at the time. We conducted initial studies of AO properties and immediately realized that this probe will have great potential for studying exocytotic processes. In this application we propose to use what we learned from our preliminary work to develop the application of AO in investigation of the kinetics of the mucus formation process. Our goal is to establish new technology for imaging cellular processes associated with mucus release with high spatial and temporal resolution. Distinctly different spectroscopic properties of the monomeric and aggregated form of AO open a novel possibility for the development of a two excitation wavelength, ratiometric, TIRF- FLIM approach that will allow, for the first time, detailed kinetics studies of mucin swelling and monitoring of its rheologic (viscoelastic) properties at the single mucin granule level. Total internal reflection fluorescence (TIRF) will allow surface confined excitation for monitoring membrane processes within a 100 nm layer. Fluorescence lifetime imaging (FLIM) will allow very precise, fast detection on the monomer-aggregate equilibrium independently of the granule size. Use of two excitation wavelengths as interleaved pulses with adjustable relative delay will allow simultaneous monitoring of monomer and aggregate populations thus highly increasing sensitivity and speed for detection.